Single stranded oligonucleotides, probes, primers and method for detecting spirochetes

ABSTRACT

The invention concerns single stranded oligonucleotides comprising a sequence of at least 12 consecutive nucleotide motifs contained in one of the sequences SEQ ID NO: 1 to SEQ ID NO: 4 wherein “n” represents an identical or different nucleotide selected among inosine or an equimolar mixture of 4 different nucleotides selected among a, t, c or g, and among oligonucleotides complementary thereto. The invention also concerns using the oligonucleotides to detect bacteria of the Spirochaetales order. The invention is based on the use of specifically defined sequences in the rpoB gene of spirochetes coding for the beta subunit of the bacterial RNA polymerase.

This invention concerns the techniques for the detection and/oramplification and sequencing, using probes or oligonucleotide primers,and the application of these probes or primers to detect or identifybacteria in the order of Spirochaetales (spirochetes).

In certain diagnostic tests, in particular during the search for a humanor animal infection in the nervous system or a search after an arthropodbite, for example, it is often necessary to obtain a fast responseconcerning the presence of bacteria, and more specifically spirochetes,in a sample. However, the techniques currently developed, such as directexamination after Gram coloration or culture often fail due to the lackof Gram colour and the lack of growth in the culture medium.

In the case of spirochetes, forming a family of bacteria including thespecies Leptospira, Borrelia, Treponema and Serpulina, responsible forinfectious diseases, such as meningoencephalitis, a culture is notpossible. Therefore, the identification of spirochetes has still notbeen solved.

Given the increase in infectious diseases over the last twenty years andthe dramatic consequences of these infectious diseases, it is necessaryto develop a fast and specific method to detect infectious pathogenicssuch as spirochetes.

A possible solution, able to palliate the impossibility of the cultureof bacteria, lies in the use of technologies related to nucleic acidsand genetic material, in particular PCR (Polymerase Chain Reaction)methods, in order to determine whether a gene, a part of a gene or anucleotide sequence is present in a living organism, a cell extract fromthis organism or a sample. Given that any gene or part of a gene ischaracterised by a specific sequence of nucleotide bases, it istherefore possible to directly search for the presence of all or part ofthe said specific sequence within a sample containing a mixture ofpolynucleotides.

Different types of methods for the detection of nucleic acids aredescribed in the literature. These methods are based on the propertiesof purine-pyrimidine pairing of complementary stands of nucleic acids inthe DNA—DNA and RNA—RNA duplex. This pairing process is carried out bythe establishment of hydrogen bonds between adenine-thymine (A-T) andguanine-cytosine (G-C) bases of the double strand DNA. Pairs ofadenine-uracile (A-U) bases may also form by hydrogen bond in theDNA-RNA or RNA—RNA duplex. The pairing of strands of nucleic acid todetermine whether a given molecule of nucleic acid is present or absentis commonly called “hybridation of nucleic acids” or simply“hybridation”.

An example of the PCR method includes the determination of a sequence onthe base of RNAr 16S. However, this method has limits due to potentialproblems of contamination that hinder the diagnosis.

To make up for this disadvantage, new genetic markers have been foundfor the specific detection of bacteria belonging to the order ofspirochetes in any sample, without a preliminary stage of bacteriaculture. These new markers or oligonucleotides are the object of theinvention.

The determination of these new markers relies on the use of specificallydefined sequences in the gene rpoB of spirochetes coding for the βsub-unit in bacterial RNA polymerase. In fact, zones that vary accordingto the bacteria family but that appear preserved among the order ofspirochetes are found on DNA coding for the β sub-unit of bacterial RNApolymerase. Therefore, this bacteria family can be distinguished fromother bacteria families. The observation that, in the said preservedzones, there are minor variations of sequences between certain speciesof spirochetes was used to develop, these markers specific to the orderof spirochetes.

According to Lazcano et al. [J. Mol. Evol. (1988) 27:365–376], the RNApolymerase are divided into two groups according to their origin, oneformed by viral RNA- or DNA-dependent RNA polymerase and the otherformed by DNA-dependent RNA polymerase of eucaryote or procaryote origin(archaebacteria and eubacteria). The DNA-dependant eubacteria RNApolymerase are characterised by a simple and preserved multimericconstitution called “core enzyme”, represented by αββ′ or “holoenzyme”represented by αββ′σ [Yura and Ishihama, Ann. Rev. Genet. (1979)13:59–97].

A great many studies have revealed the functional role, within themultimeric enzyme complex, of the β sub-unit of eubacteria RNApolylmerase. Archaebacteria and eucaryote RNA polymerase present a morecomplex structure that may include ten or even thirty sub-units [Pühleret al., Proc. Natl. Acad. Sci. USA (1989) 86:4569–4573].

The genes that code for the different αββ′σ sub-units of DNA-dependentRNA polymerase in eubacteria, respectively genes rpoA, rpoB, rpoC andrpoD, are classified in different groups including genes coding for theproteins that constitute ribosome sub-units or for the enzymes involvedin the duplication and repair of the genome [Yura and Ishihama, Ann.Rev. Genet. (1979) 13:59–97]. Certain authors have demonstrated that thenucleic sequences of genes rpoB and rpoC may be used to buildphylogenetic trees [Rowland et al., Biochem. Soc. Trans. (1992) 21:40s]to separate the different branches and sub-branches among the kingdom ofthe living.

Before describing the invention in more detail, the different terms usedin the description and claims are defined below:

-   -   “nucleic acid extracted from bacteria” refers to either the        total nucleic acid or the genomic DNA or the messenger RNA or        the DNA obtained from the reverse transcription of the messenger        RNA;    -   “nucleotide fragment” or “oligonucleotide” are two synonymous        terms indicating a sequence of DNA or RNA nucleotide motifs        characterised by an informational sequence of natural (or        modified) nucleic acids that are likely to hybrid, such as        natural nucleic acids, with a complementary or a roughly        complementary nucleotide fragment, in predetermined conditions        of strict stringence. The sequence may contain nucleotide motifs        with a different structure from that of natural nucleic acids. A        nucleotide fragment (or oligonucleotide) may, for example,        contain up to 100 nucleotide motifs. It generally contains at        least 10, and in particular at least 12 nucleotide motifs and        may be obtained from a natural nucleic acid molecule and/or by        genetic recombination and/or by chemical synthesis;    -   “a nucleotide motif” is derived from a monomer that may be a        natural nucleotide of nucleic acid where the constitutive        elements are a sugar, a phosphate group and a nitrogen base        chosen from among adenine, guanine, uracile, cytosine and        thymine; or the monomer is a nucleotide modified in at least one        of the former three constitutive elements. By way of example,        the modification may occur in the bases, with modified bases        such as inosine that can hybrid with any base A, T, U, C or G,        methyl-5-deoxycytidine, deoxyuridine,        dimethylamino-5-deoxyuridine or any other modified base able to        hybrid, or in the sugar, for example, the replacement of at        least one deoxyribose by a polyamide [P E Nielsen et al.,        Science, (1991) 254:1497–1500] or in the phosphate group, for        example by replacement by chosen esters, in particular among        diphosphates, alkyl and arylphosphonates and phosporothioates;    -   “informational sequence.” refers to any ordered sequence of        nucleotide motifs, where the chemical nature and the order in a        reference direction form information similar to that provided by        a sequence of natural nucleic acids;    -   “hybridation” refers to the process where, in appropriate        conditions, two nucleotide fragments with sufficiently        complementary sequences are able to associate by stable and        specific hydrogen bonds, to form a double strand. The conditions        for hybridation are determined by the “stringence”, that is, the        strictness of the operating conditions. The greater the        stringence, the more specific the hybridation. The stringence is        mainly a function of the composition in bases of a probe/target        duplex, as well as by the degree of mis-matching between two        nucleic acids. The stringence may also be a function of the        parameters of the hybridation reaction, such as the        concentration and type of ionic species found in the hybridation        solution, the type and concentration of denaturing agents and/or        the hybridation temperature. The stringence of the conditions in        which the hybridation reaction has to be carried out mainly        depends on the probes used. All of this data is well known and        the appropriate conditions may eventually be determined in each        case by routine experiments. In general, depending on the length        of the probes used, the temperature for the hybridation reaction        is approximately between 20 and 65° C., in particular between 35        and 65° C. in a saline solution at a concentration of about 0.8        to 1 μM.    -   a “probe” is a nucleotide fragment including, for example,        between 10 and 100 nucleotide motifs, in particular between 12        and 35 nucleotide motifs, with a hybridation specificity in        determined conditions to form a hybridation complex with a        nucleic acid with, in the present case, a nucleotide sequence        included in a messenger RNA or in a DNA obtained by reverse        transcription of the said messenger RNA, a transcription        product. A probe may be used for diagnostic purposes (in        particular capture or detection probes) or for therapeutic        purposes;    -   a “capture probe” is immobilised or can be immobilised on a        solid support by any appropriate means, for example, by        covalence, by adsorption, or by direct synthesis on a solid.        Examples of supports include microtitration plates and DNA        chips;    -   a “detection probe” may be marked by means of a marking agent        chosen for example from among the radio-active isotopes, the        enzymes, in particular enzymes able to act on a chromogenic,        fluorigenic or luminescent substrate (in particular a peroxydase        or an alkaline phosphatase), chromophoric chemical compounds,        chromogenic, fluorigenic or luminescent compounds, analogues of        nucleotide bases and ligands such as biotine;    -   a “species probe” is a probe that is able to identify the        species of bacteria;    -   a “genus probe” is a probe that is able to identify the genus of        a bacteria;    -   a “primer” is a probe including, for example, between 10 and 100        nucleotide motifs and with a hybridation specificity in        determined conditions for the initiation of an enzyme        polymerisation, for example, in an amplification technique such        as PCR, in a sequencing procedure, in a transcription method,        etc.

The first purpose of the present invention is a single strandedoligonucleotide chosen from among the oligonucleotides with a sequenceof at least 12 consecutive nucleotide motifs included in one of thesequences SEQ ID NO 1 to SEQ ID NO 4 described in the list of sequencesat the end of the description and among the complementaryoligonucleotides of these oligonucleotides.

In each of the sequences SEQ ID NO 1 to 4, the nucleotide “N” mentionedin the list of sequences at the end of the description represents anidentical or different nucleotide in each of the 3 positions where itappears and may represent inosine or an equimolar mixture of 4 differentnucleotides chosen from among A, T, C and G or, should the occasionarise, A, U, C and G.

When “N” represents a said equimolar mixture of nucleotides at a givenposition, this means that the nucleotide at the said positionindifferently represents A, T, C or G (or respectively, should theoccasion arise, A, U, C or G) and that the oligonucleotide according tothe invention more exactly consists of an equimolar mixture of 4 groupsof oligonucleotides in each the said groups “N” has a different meaningat the said given position and respectively represents in each of the 4groups A, T, C and G.

In each of the sequences SEQ ID NO 1 to 4, when in the 3 positions wherea nucleotide “N” appears, N represents a said equimolar mixture of 4nucleotides A, T, C and G (or N should the occasion arise A, U, C andG). The oligonucleotide forming the said sequence therefore represents amixture of 4³ (64) oligonucleotides.

As indicated in the definitions, the oligonucleotide according to theinvention may be in the form of an oligodeoxyribonucleotide (DNA) or anoligoribonucleotide (RNA) where, in this case, ‘T’ is replaced by “U”.

In particular, an oligonucleotide according to the present invention hasat least 12 motifs as described above and at not more then 50 motifs.Specifically, according to the present invention, an oligonucleotide hasfrom 12 to 35 motifs.

A preferred oligonucleotide has a sequence chosen from among thesequences SEQ ID NO 1 to 4, or the complementary sequences.

Sequence SEQ ID NO 1, the sequence from the primer called primer NO 1,has 20 nucleic acids. Sequence SEQ ID NO 2, the sequence from the primercalled primer NO 2, has 21 nucleic acids. Sequence SEQ ID NO 3, thesequence from the primer called primer NO 3, has 20 nucleic acids.Sequence SEQ ID NO 4, the sequence from the primer called primer NO 4,has 17 nucleic acids.

In the nomenclature referring to gene rpoB in E. COLI, the firstnucleotides in sequences SEQ ID NO 1 to 4 correspond to the followingpositions:

-   -   SEQ ID NO 1:1 730,    -   SEQ ID NO 2:2 900,    -   SEQ ID NO 3:3 700,    -   SEQ ID NO 4:3 850.

The inventors discovered and revealed that the said sequences SEQ ID NO1 to 4, as defined above, are not only consensual between thespirochetes, but that they are also specific to the family ofspirochetes and enclose a fragment of gene rpoB including a veryvariable zone where the sequence is specific to the species and thegenus serovar in a species of spirochete.

In fact, variable nucleotides in the complementary target sequences arefound in the positions corresponding to those of nucleotides “N” in thesequences SEQ ID NO 1 to 4 as a function of the species of bacteriaconsidered. However, the other nucleotides are preserved in all of thespecies of bacteria in the spirochete family. Since “N” representsinosine that may hybrid with any base, or an equimolar mixture of 4bases A, T, C and G, an oligonucleotide or a mixture of oligonucleotidesaccording to the invention always includes an oligonucleotide that canhybrid with a complementary target sequence of a spirochete bacteria.

In addition, since the sequences SEQ ID NO 1 to 4 enclose variable zoneswhere the sequences are specific for each species of bacteria in thespirochete family, sequences SEQ ID NO 1 to 4 not only allow for thepreparation of specific probes for the family of spirochetes, but alsothe detection and identification of the species of the said bacteria byPCR amplification by using the said sequences as primers.

In a mode of preparation of oligonucleotides according to the invention,“N” represents an inosine in all the positions.

In another mode of preparation, the oligonucleotide according to theinvention represents a mixture of oligonucleotides (in particular 4^(P)oligonucleotides) comprising sequences included in one of the sequencesSEQ ID NO 1 to 4, in which all of the nucleotides A, T, C and G arerepresented in each of the said positions where “N” appears (inparticular p positions, p representing a whole number).

Sequences SEQ ID NO 1 to 4 may be prepared by chemical synthesis usingfamiliar techniques described, for example, in the article by Itakura K.et al. [(1984) Annu. Rev. Biochem. 53:323].

A first application of an oligonucleotide from the invention is its useas a probe for the detection, in a biological sample, of bacteriabelonging to the order of spirochetes that include a nucleotide sequencewith at least 12 consecutive nucleotide motifs included in one of thesequences SEQ ID NO 1 to 4, and their complementary sequences. In therest of the description, such a probe will be called a “genus probe”.

According to the invention, the probes may be used for diagnosticpurposes, in the search to determine whether a target nucleic acid ispresent in a sample, according to all known hybridation techniques andin particular the so-called “DOT-BLOT” techniques involving the depositon a filter [Maniatis et al. (1982) Molecular Cloning, Cold SpringHarbor], the so-called “SOUTHERN BLOT” techniques involving DNA transfer[Southern, E. M., J. Mol. Biol. (1975) 98:503], the so-called “NORTHERNBLOT” techniques involving RNA transfer, or the so-called “sandwich”techniques [Dunn A. R., Hassel J. A. (1977) Cell 12:23]. In particular,the “sandwich” technique is used with a capture probe and/or a detectionprobe, the said probes are able to hybrid with two different regions oftarget nucleic acid, and at least one of the said probes (generally thedetection probe) is able to hybrid with a region in the target that isspecific to the species or group of species searched for, consideringthat the capture probe and the detection probe have to have at leastpartially different nucleotide sequences.

The nucleic acid to detect (target) may be DNA or RNA (one of thempossibly obtained after PCR amplification). In the case of the detectionof a double strand nucleic acid target, it is advisable to proceed withthe denaturation of the latter before the use of the detectionprocedure. The target nucleic acid may be obtained by extractionaccording to the known methods for nucleic acids from a test sample. Thedenaturation of a double strand nucleic acid may be carried out by thefamiliar methods of chemical, physical or enzyme denaturation, and inparticular by heating at an appropriate temperature above 80° C.

To use the aforementioned hybridation techniques, and in particular the“sandwich” techniques, a probe from the invention, called a captureprobe, is immobilised on a solid support, and another probe from theinvention, called a detection probe, is marked with an agent.

The examples of support and marker agent are defined above.

Another purpose of the invention is a method to determine whether atleast one spirochete is present in a sample containing or likely tocontain nucleic acids from at least one such bacteria, including thesteps consisting in putting in contact the said sample with at least onegenus probe from the invention, then to determine in a known mannerwhether a hybridation complex has formed between the said probe and thenucleic acid from the sample.

Examples of detection of the formation or lack of formation of ahybridation complex between the said probe and the nucleic acid includethe techniques described above, that is, the “DOT-BLOT”, “SOUTHERN-BLOT”and “sandwich” techniques.

In one specific use of this procedure to determine whether or not aspecies or a group of species of spirochete are present, a genus probefrom the invention and a species probe from the invention are used. Ofcourse, the said genus and species probes are able to hybrid with thenon-overlapping regions of a nucleic acid corresponding to the gene rpoBof spirochetes.

In an advantageous way, the genus probe is immobilised on a solidsupport and the species probe is marked with a marking agent.

The present invention also involves the application of the procedurefrom the invention to determine the presence of a determined spirochetespecies.

In fact, the results of the research, revealing alignments of sequencespreserved in spirochete species, according to the CLUSTAL method ofalignment [Higgins D. G. & Sharp P. M. (1989) Gene 73:237–244], the saidsequences have 1170 bases located between positions 1730 and 2900 ofgene rpoB by referring to the numbering of Escherichia coli gene rpoBATCC 25290, allow for the detection of at least one bacteria in theorder of spirochetes. The use of probes containing zones muted for aspecific species (with respect to the reference species, in this case E.coli) makes it possible to directly detect such a species. The sandwichhybridation techniques, using a combination of two probes (capture probeand detection probe), use, for example, a combination of a probespecific for the family of spirochetes and a probe specific for thespecies considered. It is also possible to use a combination of twoprobes specific for the said species. When they exist, these two probesare complementary for non-overlapping regions of the gene rpoB.

Another application of an oligonucleotide in the invention is its use asa nucleotide primer including a single stranded oligonucleotide chosenfrom among the oligonucleotides with a sequence of at least 12nucleotide motifs included in one of the sequences SEQ ID NO 1 to 4,that can be used in the synthesis of a nucleic acid in the presence of apolymerase by a known procedure, that is in the methods of amplificationusing such a synthesis in the presence of a polymerase (PCR, RT-PCR,etc.). In particular, a primer from the invention may be used for thespecific reverse transcription of a sequence of messenger RNA of atleast one species or at least a group of species of spirochetes toobtain a corresponding complementary sequence of DNA. Such a reversetranscription may be the first stage in the RT-PCR technique, thefollowing stage being the amplification of the complementary DNAobtained by PCR. It is also possible to use primers from the inventionfor the specific amplification by chain polymerisation reaction of theDNA sequence of the gene rpoB of at least one species or at least agroup of species of spirochetes.

In a specific case, since the said primer comprises an oligonucleotidefrom the invention it also includes the direction or anti-directionsequence of a promotor recognised by a RNA polymerase (for example,promotors T7, T3, SP6 [Studier F W, B A Moffatt (1986) J. Mol. Biol.189:113]. Such primers can be used in the methods to amplify nucleicacid involving a transcription stage, such as, for example, the NASBA or3SR techniques [Van Gemen B. et al. Abstract M A 1091, 7^(th)International Conference on AIDS (1991), Florence, Italy].

Another purpose of the invention is a nucleotide primer comprising asingle stranded oligonucleotide chosen from among the oligonucleotideswith a sequence of at least 12 consecutive nucleotide motifs included inone of the sequences SEQ ID NO 1 to SEQ ID NO 4 that can be used for thetotal or partial sequencing of the gene rpoB in any species ofspirochete. In particular, the nucleotide primer can be used for thesequencing of an amplified nucleic acid.

In fact, the oligonucleotide primers concerned by the invention allowfor the amplification and then the sequencing of the gene rpoB in allspirochetes, and the identification of any spirochete by bio-dataprocessing analysis of this sequence and the recognition of new andunknown species of spirochetes. The sequencing involves the acquisitionof the total or partial sequence of the gene rpoB by a familiar method,absorptive polymerisation using di-deoxynucleotides [Sanger F., CoulsonA. R. (1975) J. Mol. Biol. 94:441] or multiple hybridiations using DNAchips.

Primers NO 1 to NO 3 can be used as 5′ primers and primers NO 2 to NO 4as 3′ primers to amplify a fragment of gene rpoB enclosed in 5′ and 3′by the said primers.

In one mode, a set of two primers is used consisting of two differentoligonucleotides chosen from among the oligonucleotides with a sequenceof at least 12 nucleotide motifs included in sequences SEQ ID NO 1 to 3for 5′ primer and SEQ ID NO 2 to 4 for 3′ primer.

The present invention also includes a method, characterised in that thefragment of gene rpoB of the said bacteria is first amplified withprimers according to the invention, then the said fragment is put intocontact with a probe from the said bacteria according to the invention.Whether or not a hybridation complex is formed between the said probeand the said fragment is determined.

The present invention also involves a method to determine whether atleast one bacteria in the order of spirochaetales is present in a samplecontaining or likely to contain nucleic acids from at least one suchbacteria, characterised in that the said sample is put into contact withprimers according to the invention. Amplification is then carried outand whether or not an amplification product appears is determined.

In one mode, a method to determine whether at least one bacteria fromthe order of spirochaetales is present in a biological sample containingor likely to contain nucleic acids from the said bacteria, ischaracterised in that the method amplifies and carries out thesequencing of a fragment of gene rpoB of the said bacteria in the saidsample by means of primers according to the invention and the sequenceof the fragment of gene rpoB of the gene obtained is compared with theknown sequence of a fragment of gene rpoB of the said bacteria in asample if the sequence of the fragment obtained is identical to theknown sequence of the fragment of gene rpoB of the said bacteria.

The present invention also involves a detection probe, in a biologicalsample, specific for a species of bacteria belonging to the order ofspirochaetales, characterised in that it includes a fragment of generpoB that can be obtained by amplification by means of primers accordingto the invention, preferably with one of the sequences SEQ ID NO 1 to 3as 5′ primer and one of the sequences SEQ ID NO 2 to 4 as 3′ primer. Thesaid fragment of gene rpoB enclosed by the oligonucleotides according tothe invention actually corresponds to a very variable zone according tothe species of said bacteria belonging to the order of spirochaetalesand that is specific for each said species.

Finally, the invention also includes a gene therapy probe to treatinfections induced by at least one species or a group of species ofspirochetes, the said probe including an oligonucleotide as definedabove. This gene therapy probe, able to hybrid on the messenger RNAand/or on the genome DNA of the said bacteria, can block the translationand/or transcription and/or duplication phenomena.

The principle of the gene therapy methods is known and is mainly basedon the use of a probe corresponding to an anti-direction strand. Theformation of a hybrid between the probe and the direction strand is ableto perturb at least one of the stages in the decoding of the geneticinformation. Gene therapy probes can thereby be used as anti-bacteriadrugs, to fight infections caused by spirochetes.

The invention will now be described in detail using the followingexperimental report.

The following description will better be understood using FIGS. 1 and 2in which:

FIG. 1 is a photo of an electrophoresis gel revealing the detection ofspirochetes in a mixed bacteria suspension using the probes in theinvention; and

FIG. 2 is a photo of an electrophoresis gel revealing the specificamplification by PCR of fragments of gene rpoB of spirochetes by usingthe primers in the invention.

The strains of spirochetes used in the following examples, that isBorrelia burgdorferi, Borrelia recurrentis, Treponema pallidum,Leptospira biflexa serovar patoc (hereafter called Leptospira biflexa),Leptospira interrogans serovar icterohaemmorragiae (hereafter calledLeptospira icterohaemmorragiae) and Leptospira interrogans serovaraustralis (hereafter called Leptospira australis), have all beenobtained from the ATCC collection, except for Borrelia recurrentis thatis available at the Centre National de référence, Institut Pasteur Paris(France). The ATCC number of Borrelia burdorferi is 35210, that ofTreponema pallidum is 27087, that of Leptospira biflexa is 23582, thatof Leptospira icterohaemmorragiae is 43642 and that of Leptospiraaustralis is 23605.

The Borrelia and Leptospira strains were grown at 30° C. on BSKII andEMJH media, respectively [Barbour A. G. (1984) Yale J. Biol. Med.57:521]. Since T. pallidum can not be grown in vitro, this pathogenicbacteria was propagated by injection in the testicles of a rabbit.

The other strains of bacteria used, Escherichia coli, Staphylococcusaureus, Streptococcus salivarius and Pseudomonas aeruginosa wereclinically isolated from patients hospitalised in Marseille.

EXAMPLE 1 Specific Detection of Spirochetes Using Probes from theInvention

This experiment was carried out with the following spirochete strains:Borrelia burdorferi, Treponema pallidum, Leptospira biflexa, Leptospiraicterohaemmorragiae, Leptospira australis and Borrelia recurrentis, aswell as with Staphylococcus aureus.

A mixed bacteria suspension was prepared by mixing a spirochete DNA witha extract of Staphylococcus aureus.

A PCR method was carried out using the QIAamp tissue kit (Qiagen) andthe Gibco Polymerase Taq (Gibco BRL, USA). After a first stage ofdetermination (94° C. for 2 minutes), a cycle of 3 stages consisting of94° C. for 30 s, 52° C. for 30 s and 72° C. for 1 minute was repeated 35times. The PCR programme was terminated. The resulting amplicons werepurified and sequenced as indicated above.

The results are indicated in FIG. 1, in which:

band 1 corresponds to molecular mass markers (Boehringer),

band 2 corresponds to S. aureus alone,

band 3 corresponds to B. burgdorferi+S. aureus,

band 4 corresponds to B. recurrentis+S. aureus,

band 5 corresponds to T. pallidum+S. aureus,

band 6 corresponds to L. biflexa+S. aureus,

band 7 corresponds to L. australis+S. aureus,

band 8 corresponds to L. icterohaemmorragiae+S. aureus.

The following primers were used:

panel A: RNAr primers 16S FDI and RD3 [Weisburg et al. J. Bacteriol.(1991) 173:697–703].

panel B: the primers 1730D and 2900R from the invention.

The primer 1730D in sequence SEQ ID NO 1 (5′CTTGGNCCNGGNGGACTTTC3′) inwhich “N” is inosine and the primer 2900R has the sequence SEQ ID NO 2(5′AGAAATNAANATNGCATCCTC3′) in which “N” is inosine.

The results show that the primers in the invention were not able toamplify the DNA of S. aureus alone, and therefore detect S. aureus, butwere able to amplify that of the spiochetes, and thereby detect it, evenin the presence of S. aureus (panel B), as opposed to primers 16S thatwere able to amplify the DNA only in S. aureus (panel A).

EXAMPLE 2 Specific Amplification of Fragments of Gene rpoB Using thePrimers in the Invention

This experiment was carried out with the following spirochete strains:Borrelia burdorferi, Treponema pallidum, Leptospira biflexa, Leptospiraicterohaemmorragiae, Leptospira australis and Borrelia recurrentis, aswell as with the other non-spirochete strains Escherichia coli,Staphylococcus aureus, Streptococcus salivarius and Pseudomonasaeruginosa.

The sequence of the gene rpoB in Treponema pallidum used is thatdescribed by Weinstock et al. [(1998) The genome of Treponema pallidum:new light on the agent of syphilis. FEM Microbiol. Rev. 22:323–332].

The sequence of gene rpoB in Borrelia burgdorferi used is that describedby Alekshun et al. [(1997) Molecular cloning and characterisation ofBorrelia burgdorferi rpoB. Gene 186:227–235].

The DNA sequence of the gene rpoB of Leptospira biflexa, Leptospiraicterohaemmorragiae, Leptospira australis and Borrelia recurrentis wereobtained by PCR in the following way:

The genome DNA of Leptospira biflexa was extracted according to thestandard procedures with phenol/chloroform [Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual. Cold Spring Harbour, N.Y.]. Inthis first stage of the experiment, amplifications by PCR were carriedout with primers SEB1 (5′-AAC CAG TTC CGC GTT GGC CTG G-3′) and SEB2(5′-CCT GM CM CAC GCT CGG A-3′) (SEB for Staphylococcus aureus,Escherichia coli and Borrelia subtilis) and the polymerase DBA Taq byEurogentec (Seraing, Belgium) by using 5 μl of DNA for a final volume of50 μl. After a first stage of denaturation (95° C. for 1.5 min), a cyclewas repeated 35 times consisting of 3 stages of 95° C. for 20 s, 50° C.for 30 s and 72° C. for 1 min. The PCR programme was completed with asingle stage of extension of 3 min at 72° C. (Peltier PTC 200 heat cyclemodel, MJ Research, Watertown, Mass., USA).

The amplicons obtained by electrophoresis were then separated on 1%agarose gel and they were visualised by coloration with ethidiumbromide.

The sequencing of the gene was then carried out by pruification of thesamples with the PCR QIAquick purification kit (Qiagen, Hilden, Germany)and they were allowed to react with the dRhodamine Terminator CycleSequencing Ready Reaction buffer (DNA sequencing kit, Perkin-Elmer).Finally, the electrophoresis of the reactive products was carried outwith the automatic Applied Biosystems DNA sequencor model ABI 310(Perkin-Elmer).

The sequence of the 5′ end of gene rpoB was obtained by using aUniversal Genome Walker™ kit (Clontech, Palo Alto, Calif., USA) in thefollowing manner. Five groups of genome DNA fragments called GenomeWalker “banks” were built by using DraI, EcoRV, PvuII, ScaII and StuIrestriction enzymes. These banks were used for the prior sequencing ofthe genome DNA by using a specific primer for the gene whose sequencecorresponds to the region as close as possible to the known 5′ end ofthe gene and an adaptation primer supplied by the kit.

The amplified fragments were purified and a source of DNA was used for asecond enclosed PCR. The amplicons obtained were then treated asdescribed above for the automatic sequencing.

Gene rpoB from the Leptospira biflexa obtained has the sequence SEQ IDNO 5 and has 3876 nucleic acids. Gene rpoB from the Leptospiraicterohaemorragia obtained has a sequence SEQ ID NO 6 and has 914nucleic acids. Gene rpoB from the Leptospira australis obtained has thesequence SEQ ID NO 7 and has 949 nucleic acids. Finally, gene rpoB fromthe Borrelia recurrentis obtained has the sequence SEQ ID NO 8 and has800 nucleic acids.

The DNA sequences of the gene rpoB from Escherichia coli, Staphylococcusaureus, Streptococcus salivarius and Pseudomonas aeruginosa aredescribed in the literature (Gene Bank).

The fragments of gene rpoB to amplify consisted of 949 pb.

The amplifications of the fragments of gene rpoB by PCR were carried outas in example 3, except that each species was used separately.

The results are indicated in FIG. 2 where:

band 1 corresponds to the molecular mass markers (Boehringer),

band 2 corresponds to B. burgdorferi,

band 3 corresponds to B. recurrentis,

band 4 corresponds to T. pallidum,

band 5 corresponds to L. biflexa,

band 6 corresponds to L. australis,

band 7 corresponds to L. icterohaemmorragiae,

band 8 corresponds to E. coli,

band 9 corresponds to S. aureus,

band 10 corresponds to Str. salivarius,

band 11 corresponds to Ps. aeruginosa,

band 12 corresponds to a negative reference without DNA.

The following primers were used:

panel A: primer 1730D and primer 2900R from the invention,

panel B: primer 1730D and primer 3800R from the invention and

panel C: RNAr primers 16S RD1 and FD3.

Primer 3800R has the sequence SEQ ID NO 4 (5′GCTTCNAGNGCCCANAC3′) inwhich “N” is an inosine.

The results show that the primers in the invention only allowed for theamplification of the DNA fragments of spirochetes (panels A and B) whileprimers 16S amplified everything (panel C).

The primer SEQ IS NO 3 (5′GGGTGNATTTTNTCATCNAC3′) may also be used as aprimer. Primers NO 1 and 3 according to the invention allowed for theamplification and then sequencing of the amplified product, thedetection and identification of the bacteria Borrelia duttonii in whichan African tick collected in ZAIRE of the species ornithodoros moubataand the total blood of a Zaire patient presenting a recurrent fever.

1. An isolated oligonucleotide consisting of a sequence selected fromthe group consisting of SEQ ID NO: 1 to SEQ ID NO: 4 and sequencescomplementary thereto, wherein “n” represents inosine.
 2. A mixture of64 isolated oligonucleotides each consisting of a sequence selected fromthe group consisting of SEQ ID NO: 1 to SEQ ID NO: 4 and sequencescomplementary thereto, wherein “n” represents an equimolar mixture ofthe nucleotides a, t, c and g.
 3. A method to determine whether bacteriabelonging to the order of Spirochaetales is present in a samplecontaining or likely to contain nucleic acid from said bacteria,comprising: (a) contacting said sample with at least one probecomprising an oligonucleotide according to claim 1; and (b) determiningwhether a hybridation complex forms between said at least one probe andsaid nucleic acid in said sample.
 4. A method according to claim 3,wherein said oligonucleotide is immobilised on a solid support.
 5. Amethod according to claim 3, wherein said oligonucleotide is marked witha tracing agent.
 6. A method to determine whether bacteria belonging tothe order of Spirochaetales is present in a sample containing or likelyto contain nucleic acid from said bacteria, comprising: (a) contactingsaid sample with at least one probe comprising a mixture ofoligonucleotides according to claim 2; and (b) determining whether ahybridation complex forms between said at least one probe and saidnucleic acid in said sample.
 7. A method according to claim 6, whereinsaid oligonucleotides are immobilised on a solid support.
 8. A methodaccording to claim 6, wherein said oligonucleotides are marked with atracing agent.
 9. A method to determine whether bacteria belonging tothe order of Spirochaetales is present in a sample containing or likelyto contain nucleic acid from said bacteria, comprising: (a) contactingsaid sample with primers comprising oligonucleotides according to claim1; (b) carrying out an amplification; and (c) determining the presenceor absence of an amplification product.
 10. A method to determinewhether bacteria belonging to the order of Spirochaetales is present ina sample containing or likely to contain nucleic acid from saidbacteria, comprising: (a) contacting said sample with primers comprisingmixtures of oligonucleotides according to claim 2; (b) carrying out anamplification; and (c) determining the presence or absence of anamplification product.
 11. A method according to claim 9 or 10, furthercomprising: (d) sequencing the amplified fragment when an amplificationproduct is obtained; and (e) comparing the sequence of the amplifiedfragment with the known sequence of gene rpoB of the bacteria, whereinthe species of the bacteria is determined if the sequence of theamplified fragment is identical to that of the known sequence.